The speed of fermentation of sugars to ethanol is a function of 1) yeast concentration, 2) basic composition of the fermentation media, and 3) levels of nutrients, pH and temperature. In general, fermentation rates increase linearly with increasing cell density.F=X*ν(S, P, N, T)  Eq. 1where                F is ethanol productivity as g ethanol produced per liter per hour,        X is g cells per liter, and        ν(S, P, N) is the specific ethanol production rate of ethanol (P), as a function of substrate (S), product concentration (P), nutrients (N), and temperature (T).        
Dale et al (1994) showed that the inhibitory effects of high concentrations of 1) substrate, 2) product, and 3) salts can be linked to a single solution property, osmolality, which is basically the osmotic potential of the solution. Thus Equation 1 can be more accurately written as
 F=X*ν(Ost, N, T)  Eq. 2
where Ost is total solution osmolality which is in general an additive property of the osmolality of the various components in the mediaOst=Os(S)+Os(P)+Os(I)  Eq. 3
where Os(S), Os(P), Os(I) are the osmolality due to the of the substrate (sugar, S), product (ethanol, E) and inerts/salts (I) in the media, respectively Dale et al, (1994) developed an osmolality describing both substrate and product inhibition of the ethanolic fermentation as:ν=νm[1−Ost—/kvm]  Eq. 4μ=μm[1−Ost—/kvm]  Eq. 5Growth (μ) is more strongly inhibited by osmolality than is productivity (ν) with kνm values of ranging from around 2 to 2.5 os/kg, while kνm runs 3.5 to 5.0 depending upon yeast species, osmo-tolerance, and ethanol tolerance. Dale et al, 1994, show that for a standard Saccharomyces strain studied by Letourneau and Villa, 1987, that growth rates are totally inhibited at an osmolality of 1.9, while productivity is totally inhibited at 4.2. Equation 7 allows one to calculate the combined effects of sugars, ethanol, and salts on the fermentation rates. Cane and beet molasses are characterized by a high level of salts or minerals. The osmotic effect of these salts can be combined by lumping the salts as a single osmotic group forming 11% of the solids in an 80% molasses. The data are based on molasses osmolality by fitting the simple, lumped equation Eq. 3 with excellent results, as shown in FIG. 3. Using vinasse recycle reduces liquid effluent from the fermentation process, but causes the salts to build up in the fermentation broth, increasing solution osmolality, and inhibiting the fermentation rates as shown in Table 5. Thus, the ethanol producer must balance or optimize the benefits of reduced effluent waste water with the somewhat slower fermentation rates obtained.
Increasing temperature generally speeds a fermentation until the temperature becomes high enough to cause cell death. Fermentation rates are generally noted to increase from 20° to 32° C., doubling with a 5° C. increase in temperature.
There are two basic methods for accelerating the fermentation rates of a sugar media to ethanol, 1) increase the cell density, and/or 2) reduce the concentration of inhibitory compound(s) (with ethanol being most inhibitory due to its osmolality and toxic effects) in the media as suggested by Dale in prior U.S. Pat. Nos. 4,665,027 and 5,141,861. The focus of the invention described here is a method for maintaining a very high active cell density in the bio-reactor through the development and maintenance of a dense ‘flocculated’ yeast solid phase in the reactor. During a normal batch ethanol fermentation with standard S. cerevisae strains, a final cell concentration of between 1.5 and 15 g/l cells is achieved. It is often noted that cell growth completely stops after a certain cell density is reached (Holzberg et al, 1967). The oxygen tension in the fermentation is important in these batch fermentations, as the cells will convert a larger faction of the sugar substrate towards cell mass production as the amount of oxygen available to the cells increases. Trace oxygen can serve as a nutrient during the anaerobic fermentation of sugars, allowing the fermentation rate to increase with more cells produced. Cysewski and Wilke (1978) show an optimal oxygen tension of about 0.1 mm O2. To maintain a cell density higher than the natural maximum attained in the fermenter, methods for keeping the cells in the fermenter must be utilized. A high cell density can be maintained either by recycling cells (through membrane or centrifugal techniques) or by retaining or immobilizing the cells within the reactor. Immobilization would seem to be advantageous as the capital expense of a cell recovery and recycle system can be eliminated. There has been a good deal of work over the last 10-15 years on immobilizing organisms to maintain a high cell density in the bioreactor. Immobilization can take one of several approaches, 1) entrapment within a gel bead or plate, 2) adsorption onto a solid matrix, or 3) self-agglomeration or flocculation into flakes or pellets.
Dale & Perrin, 1994, showed good performance of a yeast immobilized on a absorbent matrix. By incorporating simultaneous gas stripping of the ethanol product, we were able to ferment very concentrated molasses feed. Fermentation rates, F, of 12 to 65 g ethanol/L hr were noted on a liquid hold-up volume basis (about 4 to 21 g/L hr on a total reactor volume basis).
The use of flocculent yeast flakes or pellets to speed fermentation has been suggested by several researchers. Some yeasts have the property of joining together in clumps or flocs, with these multi-cell clumps having a much more rapid settling velocity than single cells. Flocculation is an important factor in the brewing process. After a potable beer or wine has completed its fermentation, it is desirable to have the yeast settle out. Standard S. cerevisae used for beer and wine fermentations is selected to have this post-fermentation flocculation characteristic. Standard wine or champagne yeasts settle over a period of 150 to 300 minutes if there is no fermentation activity to suspend the cells (Arikan and Ozilgen, 1992), while flocculent cells tend to settle so quickly it is difficult to get an OD on the cells as the cells settle in one minute in a cuvette (Castellon and Menawat, 1990). There is a body of literature on flocculation available with a review of the literature available (Calleja, 1989) and a number of papers discussing the effects of sugars (Kihn et al, 1988), ions [specifically sodium as a deflocculant (Castellon and Menawat, 1990) and calcium as a pro-flocculant (Kihn et al, 1988a; 1988b; and Masy et al, 1990)]. A microscopic study of flocculating fission yeast was reported by Sowden and Walker (1987, 1989) where a “hairy” or “mucilaginous” coating of the yeast is described. Soares et al, 1992, show that cell-cell interaction is important with a cell suspension of 2×107 cell/ml reaching a free cell density of 0.5×106 cell/ml in 2 minutes while a suspension starting at 1×107 cell/ml also settled to this same free cell density in 2 minutes. Faber et al (U.S. Pat. No. 4,567,145) describe a respiration deficient, flocculent strain of S. uvarum which, when settled externally to the reactor and then recycled to a single fermenter, could produce 5 to 7% ethanol at a fermentation rate of 50 g/L hr on a glucose feed.
Dale et al (1985) showed that, for an immobilized cell population exposed to constant conditions of ethanol and sugar, the steady state live cell fraction can be estimated based on a number of simplifying assumptions as:Xssl=[μ/(μ+Kd)]  Eq. 6Where: μ is the specific growth rate of the cells (hr−1),and Kd is the death rate constant for the yeast (hr−1) (where Kd is a function of temperature, nutrient concentration, and toxic/inhibitory compound concentrations). Based on this analysis, we can see that if a cell population (i.e. the cells in one particular yeast pellet) is exposed to continuous conditions of zero growth, the steady state live cell density will be zero. Thus it is important for a pellet to occasionally see conditions allowing cell growth. There will be no problem for operation of reactor in the consecutive batch mode, as conditions at the beginning of the fermentation are conducive to growth (low ethanol, fresh nutrients). For the continuous cascade reactor, it is important that stage 1 conditions be maintained such that there is cell growth, with the overflow of these younger cells from stage one to stage two and subsequent stages refreshing the population of these stages where there will be little or no cell growth due to the higher solution osmolality (largely due to ethanol concentration).
The use of highly floccculent yeast for continuous reactors has been demonstrated by APV in a tower fermenter. Cell densities of 70-80 g/l were reported (Greenshield and Smith, 1970). Chen and Gong (1986) did further work on a flocculent tower type reactor. Cysewski and Wilke (1977) used a simple settler with cell recycle to attain high cell densities in a stirred reactor. However, no one has successfully applied a flocculent yeast fermentation to molasses, shown the stable long term performance obtained with our strain of yeast, or developed a minimal effluent process as described in this invention.
According to C. Kurtzman & J. W. Fell, “The Yeasts: A Taxonomic Study”, Elsevier Press, 1998, the characteristics of Saccharomyces cerevisae are as follows:
Fermentative (anaerabobic conversion to ethanol)Glucose+Galactosev (variable)Sucrose+MaltosevLactose−Raffinose+Trehalose−MelibiosevStarch−AssimilationGlucose−Galactosev (variable)Sorbose−Sucrose+Maltose+Cellobiose−Trehalose+Lactose−MelibiosevRaffinose+MelezitosevInulin−Soluble Starch−D-xylose−L-Aribinose−D ribose−L-Rhamnose−D-Glusoamine−N Aceyl-D-glucosamine−Methanol−Ethanol+Glycerol−Erythritol−Ribitol−Glacatitol−D Mannitol−D-Glucitol−Methyl glucosidevSalicin−D GluconatevD-LactatevSuccinatevCitrate−Inositol−Hexadecane−Nitrate−Vitamin free−Growth above 30 C+Growth above 37 CvDiazonium blue B reaction is negative
Strain BPSC-15 is distinguished from other known strains in so far as vegetative reproduction by multi-lateral budding is characterized by spherical cells, with no generation of mycelia. Yeasts remain attached in clumps formed with thousands of cells per clump.
A summary of distinguishing characteristics of BPSC-15 from its parent strain is provided by the following table:
ParentBPSC 15Strain1.Highly Flocculentpospos2.Fermentation @ 5.0 Os/Kg Osmolalityposneg3.Fermentation @ 3.8 Os/Kgpospos4.Fermentation of Glucose Fructose,posposSucrose to Ethanol5.Long Term stability of 1-3 mm floc pelletsposneg6.Long Term stability of floc pellets inposnegunfiltered molasses media
Strain BPSE-15 was deposited at ARS Patent Culture Collection “NRRL” under the Budapest Treaty on Oct. 25, 2002 and accorded Accession No. NRRL Y-30630. The depository is the National Center for Agricultural Utilization Research, Agricultural Research Service, USDA, 1815 North University Street, Peoria, Ill. 61604-3999 U.S.A.